The present application relates to a microparticle measuring apparatus and, more particularly, to a microparticle measuring apparatus capable of highly accurate measurement which has its optical axis corrected at arbitrary or prescribed timing during measurement.
There has been an apparatus used to optically identify the characteristic properties of such microparticles as those associated with living bodies (e.g., cells, microorganism and liposomes) and with industries (e.g., latex particles, gel particles, and synthetic particles). It is so designed as to introduce a dispersion of microparticles into a flow channel and direct a light beam to the microparticles passing through the flow channel.
Most popular among apparatuses to measure microparticles associated with living bodies is one for flow cytometry, which is called flow cytometer. (See “Saibou Kougaku (Cell Engineering), supplement volume, Experiment Protocol Series, Mastering of Flow Cytometry,” by H. Nakauchi, issued by Shuujunsha, 2nd edition, issued Aug. 31, 2006.) One type of it is intended only to identify the characteristic properties of microparticles and the other is so designed as to fractionate microparticles with desired properties according to the results of measurement obtained by the first type. The latter, which is used to fractionate cells, is referred to as “cell sorter.”
The existing flow cytometer is so designed as to determine the characteristic properties (e.g., size and structure) of such microparticles as cells and microbeads in the following manner. A sample solution containing microparticles of interest is introduced into the center of the laminar flow of a sheath solution passing through a flow cell, so that the microparticles are lined up in the flow cell. The microparticles passing in a line through the flow cell are illuminated with a laser beam, and the scattered light or fluorescent light emanating from them is detected for determination of their characteristic properties. The system for directing a laser beam to the sample flow in the optical detection unit is divided into “closed system” (which is designed such that illumination with a laser beam is accomplished in the flow cell) and “jet-in-air system” (which is designed such that a laser beam is directed to the jet water column discharged from a jet nozzle). The foregoing step may optionally be followed by fractionation of microparticles having desired characteristic properties in such a way that the sample solution containing microparticles is discharged in the form of droplets from the flow cell and individual droplets are moved in different controlled directions.
Japanese Patent Laid-open No. 2007-46947 discloses a existing cell sorter (as shown in its FIG. 7) which is composed of a flow cell having a flow channel that causes cells (dyed with a fluorescent labeling reagent) to be lined up therein, an optical system that illuminates the cells with a laser beam and detects scattered light or fluorescent light, and a cell fractionating system that controls the moving direction of droplets discharged out of the flow cell. This cell sorter is provided with the optical detection unit of closed system.
For the optical detection unit to be capable of efficient detection of scattered light or fluorescence emanated from microparticles, it needs adjustment for the laser beam to orthogonally intersect with and focus on the sample flow. This step is usually called optical axis correction or “calibration.” The optical axis correction is accomplished by flowing microbeads for calibration and adjusting the position and focus of the condenser lens while watching the histogram data of such microbeads. In this way the relative position of the laser beam, the sample flow, and the detector is optimized. Japanese Patent Laid-open No. Hei 11-83724 and Japanese Patent Laid-open No. Hei 9-196916 disclose the microbeads for calibration used for the optical axis correction.
In the meantime, there has recently been developed a microchip which is composed of a silicon or glass substrate and a region or flow channel formed therein in which chemical or biological analysis is carried out. The analytical system using such a microchip is referred to as μ-TAS (micro-total-analysis system) or lab-on-chip or biochip.
The μ-TAS may be applied to the technology for fractionation of microparticles which examines microparticles for their characteristic properties by optical, electrical, or magnetic device while they are passing through the flow channel or region formed in the microchip. For example, Japanese Patent Laid-open No. 2003-107099 discloses a microchip for microparticles separation which is composed of a substrate and those components formed therein as listed below. A flow channel for introduction of a solution containing microparticles. A flow channel to form a sheath flow therein which is arranged along at least one side of said first flow channel. A microparticle measuring unit to measure the thus introduced microparticles. Two or more microparticle fractionating flow channels to fractionate and recover microparticles which are arranged downstream said microparticle measuring unit. The foregoing microchip has an electrode near the entrance of the microparticle fractionating flow channel from the microparticle measuring unit. The microparticle fractionating apparatus provided with this microchip is able to control the direction of movement of microparticles by mutual action between the electric field of the electrode and the microparticles, thereby fractionating microparticles.
The flow cytometer (of microchip type) based on μ-TAS may have the flow channel formed in a disposable microchip so as to prevent cross-contamination of samples during measurement.